Phosphatidic acid species 34:1 mediates expression of the myo-inositol 3-phosphate synthase gene INO1 for lipid synthesis in yeast

Depletion of exogenous inositol in yeast results in rising levels of phosphatidic acid (PA) and is correlated with increased expression of genes containing the inositol-dependent upstream activating sequence promoter element (UASINO). INO1, encoding myo-inositol 3-phosphate synthase, is the most highly regulated of the inositol-dependent upstream activating sequence–containing genes, but its mechanism of regulation is not clear. In the current study, we determined the relative timing and kinetics of appearance of individual molecular species of PA following removal of exogenous inositol in actively growing wild type, pah1Δ, and ole1ts strains. We report that the pah1Δ strain, lacking the PA phosphatase, exhibits a delay of about 60 min in comparison to wildtype before initiating derepression of INO1 expression. The ole1ts mutant on the other hand, defective in fatty acid desaturation, when grown at a semirestrictive temperature, exhibited reduced synthesis of PA species 34:1 and elevated synthesis of PA species 32:1. Importantly, we found these changes in the fatty acid composition in the PA pool of the ole1ts strain were associated with reduced expression of INO1, indicating that synthesis of PA 34:1 is involved in optimal expression of INO1 in the absence of inositol. Using deuterium-labeled glycerol in short-duration labeling assays, we found that changes associated with PA species 34:1 were uniquely correlated with increased expression of INO1 in all three strains. These data indicate that the signal for activation of INO1 transcription is not necessarily the overall level of PA but rather levels of a specific species of newly synthesized PA 34:1.

Maria Laura Gaspar* , Manuel A. Aregullin, Yu-Fang Chang, Stephen A. Jesch, and Susan A. Henry From the Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA

Edited by Dennis Voelker
Depletion of exogenous inositol in yeast results in rising levels of phosphatidic acid (PA) and is correlated with increased expression of genes containing the inositoldependent upstream activating sequence promoter element (UAS INO ). INO1, encoding myo-inositol 3-phosphate synthase, is the most highly regulated of the inositol-dependent upstream activating sequence-containing genes, but its mechanism of regulation is not clear. In the current study, we determined the relative timing and kinetics of appearance of individual molecular species of PA following removal of exogenous inositol in actively growing wild type, pah1Δ, and ole1 ts strains. We report that the pah1Δ strain, lacking the PA phosphatase, exhibits a delay of about 60 min in comparison to wildtype before initiating derepression of INO1 expression. The ole1 ts mutant on the other hand, defective in fatty acid desaturation, when grown at a semirestrictive temperature, exhibited reduced synthesis of PA species 34:1 and elevated synthesis of PA species 32:1. Importantly, we found these changes in the fatty acid composition in the PA pool of the ole1 ts strain were associated with reduced expression of INO1, indicating that synthesis of PA 34:1 is involved in optimal expression of INO1 in the absence of inositol. Using deuteriumlabeled glycerol in short-duration labeling assays, we found that changes associated with PA species 34:1 were uniquely correlated with increased expression of INO1 in all three strains. These data indicate that the signal for activation of INO1 transcription is not necessarily the overall level of PA but rather levels of a specific species of newly synthesized PA 34:1.
The anionic phospholipid phosphatidic acid (PA) is both a key precursor in lipid biosynthesis and a signaling molecule. In yeast, the level of PA in the endoplasmic reticulum (ER) plays a central role in the transcriptional regulation of lipid metabolism via the Opi1/Ino2-Ino4 (Henry) regulatory circuit (1)(2)(3)(4)(5)(6)(7)(8) (Fig. 1). PA is synthesized de novo via acylation of glycerol-3-P catalyzed by two fatty acyl CoA-dependent reactions as well as by turnover of individual phospholipids, via phospholipase D (9,10). Cellular levels of PA are impacted by its rate of synthesis and by its rate of consumption as the central precursor to both phospholipids and the neutral lipids, diacylglycerol (DAG) and triacylglycerol (TAG) (4,5,9,11). In growing cultures, PA is both rapidly synthesized and continuously converted into other lipid classes. Thus, despite its high rate of synthesis during active growth, the overall cellular levels of PA remain quite low amounting to about 1 to 3% of total cellular lipid content in wild type cells (12,13). However, in cultures adapted to growth in synthetic medium in the absence of inositol, PA levels are significantly higher than those of cells grown in its presence (5,13).
The Opi1 repressor, a soluble transcription factor that binds to both PA and the integral membrane protein Scs2 in the perinuclear ER (1,5,6), plays a central role in transcriptional regulation of INO1 and other inositoldependent upstream activating sequence (UAS INO )-containing genes (1,4,(14)(15)(16) (Fig. 1). In cells adapted to growth in the absence of inositol, phosphatidylinositol (PI) synthesis is dependent on de novo synthesis of inositol by the soluble enzyme myo-inositol 3-phosphate synthase encoded by the INO1 gene (1,4,6,17). Under these conditions, PI synthesis is reduced, and Opi1 remains associated with both PA and Scs2 in the ER allowing expression of INO1 and other UAS INO -containing genes (Fig. 1). However, when inositol is abruptly added to cultures adapted to growth in the absence of inositol, PA levels drop precipitously as PI synthesis increases (1,5). In vitro studies have revealed that Opi1 binds preferentially to PA over phosphatidylserine (PS) and to PA species in liposomes that contain saturated and shorter chain fatty acids (18). Moreover, the selective mechanism that enables binding of PA to Opi1 in preference to PS in vitro has been shown to involve a specific amphipathic helix spanning amino acids 109 to 138 (19).
While the interaction of Opi1 repressor with PA in the ER is vital to its cellular localization and to regulation of INO1 and other UAS INO -containing genes (1,5), the pool of PA in the ER consists of a range of PA species of diverse acyl-chain composition. Moreover, Pah1, a cytoplasmic Mg 2+ -dependent PA phosphatase that catalyzes the dephosphorylation of PA yielding DAG and P i (20,21), also plays a vital role in the relative synthesis of phospholipids versus DAG and TAG. Pah1 is recruited to the perinuclear ER when PA levels are elevated (22) through a process of phosphorylation and dephosphorylation. DAG generated by this reaction contributes primarily to the synthesis of TAG (21), as well as phosphatidylethanolamine and phosphatidylcholine (PC) via the Kennedy pathway (23,24). However, the specific roles of individual PA species in transcriptional regulation of INO1 and other UAS INO-containing genes have not been previously determined in vivo.
To gain insight into the potential roles of individual PA species in this complex metabolism and regulation process, we compared the overall kinetics of changes in levels of individual PA species with the kinetics of INO1 derepression in both wild type and pah1Δ cells. We found that the kinetics of distribution of deuterium-labeled glycerol into PA species 34:1 in both strains was distinctly different from that observed in the overall cellular pool of PA. Moreover, PA species 34:1 was highly correlated with the kinetics of INO1 expression in each of the two strains. More importantly, increased synthesis of PA species 34:1 was observed to precede the maximal induction of INO1 expression in both wildtype and pah1Δ cells.

Results
The kinetics of INO1 expression following removal of inositol in pah1Δ cultures differ from those in the wildtype strain In wild type yeast cells, changes in the levels of PA occurring in the course of lipid metabolism during active growth provide a metabolic signal or signals regulating expression of many genes involved in phospholipid biosynthesis (1,4,5,9). For purposes of the current investigation, the level of INO1 expression in wild type cells growing at steady state in I + medium was assigned a value of one unit ( Fig. 2A time zero). As previously reported (5), following the shift of wildtype cells to I − medium, both cellular PA content and INO1 expression increased rapidly during the first 60 min. The most rapid increase in INO1 expression occurred between 60 and 120 min following the shift to I − medium, lagging by about 60 min behind the increase in PA levels ( Fig. 2A). However, while PA levels remained elevated in wild type cells for the entire time course, INO1 expression leveled off to about 100-fold over the fully repressed state, between 300 and 420 min in the absence of exogenous inositol, as cells adapted to de novo synthesis of inositol ( Fig. 2A).
Cultures of pah1Δ exhibited about 20-fold higher expression of INO1 than wild type grown under the same conditions  Cells were grown in I + medium and phospholipids were labeled with 20 μCi/ ml [ 32 P]-orthophosphate to steady-state. At the mid-logarithmic phase of growth (A 600 = 0.5), cells were filtered, resuspended in medium lacking inositol maintaining constant the specific activity of the label, and aliquots were taken at the indicated times. Data are expressed as counts of radiolabel 32 P incorporated into total PA per OD units in the cell culture. For the analysis of INO1 expression, cells were grown and harvested at the same time points as described above for the labeling of phospholipids. Total RNA was isolated and analyzed by RT-PCR as described in Experimental procedures. Panel A (wt); panel B (pah1Δ). Data are expressed as mean ± S.D. (n = 3), **p < 0.005 and *p < 0.05 versus time zero. PA, phosphatidic acid. (Fig. 2B). Moreover, after the shift to I − medium, the kinetics of INO1 derepression in pah1Δ cultures were strikingly different from that observed in wild type cells. Initially, INO1 derepression in pah1Δ cells lagged by about 60 min in comparison to wild type under identical conditions (Fig. 2B). In addition, the level of INO1 expression achieved in the pah1Δ strain by 180 min following the shift to I − medium was only about half of that observed in wild type (Fig. 2, A and B). However, INO1 expression in pah1Δ cells increased sharply after 120 min in I − medium, reaching maximum expression at about 300 min (Fig. 2B). By 420 min in inositol-free medium, INO1 expression, in both pah1Δ and wild type, had declined to similar levels of about 100-to 150-fold over fully repressed levels (compare Fig. 2, A and B).
The shift of actively growing wild type cells from inositol containing to inositol free media induces dramatic changes in the fatty acid species composition of both membrane phospholipids and neutral lipids Consistent with data obtained previously by labeling of total phospholipids to steady state with [ 32 P]-orthophosphate in actively growing wildtype cultures (5,11), mass spectrometry analysis revealed a decrease in the levels of all molecular species of PI following the shift of wild type cells to I − medium (Fig. 3A). The major PI species, detected in wild type cells growing in I + medium (Fig. 3A, time 0), were 32:1, 34:1, and 36:1. PI species 32:1 and 34:1 declined precipitously during the first 60 min after the shift to I − medium in wild type cells (Fig. 3A), leveling off at about 180 min following the shift to I −  (Fig. 3D). Subsequently, PC species 32:1 and 34:1 are derived from DAG species 32:1 and 34:1. These PC species increased during the first 180 min (Fig. 3B) mirroring the decline seen for PI species with the same acyl chain composition (Compare Fig. 3, A and B). Since the synthesis of PI is reduced in the absence of inositol, the increase in PC species 32:1 and 34:1 suggests that excess PA species 32:1 and 34:1, normally used for PI biosynthesis, are being diverted into increased PC synthesis. As expected, TAG species were also impacted by the shift to I − medium. The most abundant TAG species in cells grown in I + medium (Fig. 4) were TAG species 48:3 (containing three C16:1 acyl chains) followed by TAG species 48:2 (containing one C16:0 acyl chain and two C16:1 acyl chains) and TAG species 48:1 (containing two C16:0 acyl chains and one C16:1 acyl chain) (Fig. 4). Following the shift to I − medium, levels of TAG species 48:3 dramatically increased from 60 to 300 min ( Fig. 4). TAG species 48:2 and 48:1 also significantly increased but to a lesser extent (Fig. 4). More importantly, evidence presented here indicates that TAG is accumulating in wild type cells upon inositol depletion. As shown above, the absence of inositol in the media altered PI metabolism significantly resulting in increased levels of PA. Under these circumstances, PA is in part a reservoir of fatty acids for the synthesis of TAG. Together, these results suggest that synthesis of storage lipids and membrane-forming lipids are interdependent and operate in a coordinated fashion to maintain lipid homeostasis in yeast cells.
Differential synthesis of PA species in response to inositol depletion in wild type and pah1Δ Since PA is precursor to both membrane forming phospholipids and the neutral storage lipid, TAG, via DAG, we initially hypothesized that the decline in PA levels in wild type cells between 120 and 240 min of growth in I − medium ( Fig. 2A) might be due to increased consumption of DAG, as precursor to the synthesis of TAG (Fig. 4). However, DAG also serves as a precursor to PC synthesis (Fig. 3B). To test this idea, we used the dga1Δ lro1Δ are1Δ are2Δ strain, defective in synthesis of the neutral lipids DAG and TAG, and a quintuple mutant strain (dga1Δ lro1Δ are1Δ are2Δ pct1Δ), lacking the ability to synthesize TAG and PC through the CDP-choline pathway. Thus, if the hypothesis that preventing the flow of PA into the synthesis of TAG and PC was correct, the collective mutations in the quintuple mutant should have resulted in higher INO1 expression following the shift to I − medium. However, contrary to this hypothesis, we found that the quintuple mutant exhibited kinetics of INO1 derepression and attenuation quite similar to those observed in wildtype cells. These results suggest that the pattern of INO1 derepression and attenuation in wild type cells, during the time course following the shift to I − medium ( Fig. 2A), does not respond to the pool of PA used to synthesize TAG and PC via the CDP-choline pathway. Instead, the distinctive kinetics of INO1 derepression between wild type and pah1Δ may be responding to the rate of PA synthesis and its short-term retention prior its utilization as a precursor in the synthesis of other lipids. To assess the relative rate of PA synthesis in the wild type and pah1Δ strains, cells were maintained in logarithmic phase and pulse-labeled with [ 32 P] orthophosphate for 25 min at 0, 60, 180, and 300 min, following the shift to I − medium (Fig. 5). Strikingly, under these conditions, wild type cells incorporated and/or retained significantly less 32 P in PA than that observed in the pah1Δ strain, especially at 180 min (Fig. 5). Within 60 min following the shift to I − medium, 32 P label associated with PA increased in both strains by about 4-fold. As expected, the level of 32 P associated with PA in wild type declined steadily from 60 to 300 min after the shift (Fig. 5). However, due to the absence of the Pah1 phosphatase, the level of 32 P associated with PA in pah1Δ remained proportionally higher than in wild type in each of the time pulses (Fig. 5). Also, consistent with the loss of the Pah1 PA phosphatase, 32 P associated with PA in the pah1Δ strain was initially higher even at 0 min after the shift, in contrast to wild type, and failed to decline in the interval between 60 and 300 min after the shift to I − medium (Fig. 5). Overall, however, these data indicate that the increase in the proportion of label incorporated into PA in both wild type and pah1Δ strains preceded the maximal induction of INO1 (Compare Figs. 2, A and B and 5).
The substantial increase in the amount of 32 P label associated with PA between 0 and 60 min in wild type cells following the shift to I − medium (Fig. 5), suggested that the increase in PA synthesis may reflect the increase of PA species 32:1 and 34:1 observed in Figure 3B. To investigate the relative rates of synthesis of individual PA species over a period of 300 min correlated with INO1 expression, we used deuterium-labeled glycerol to perform short time labeling experiments at various time points following a shift to I − medium. As shown in Figure 6, both, wild type and pah1Δ cells incorporated deuterium-labeled glycerol primarily into PA species 32:1, 34:1, and 34:2. However, in contrast to wild type, a continuous increase in label associated with PA species 32:1 and 34:2 was observed in pah1Δ cells (Fig. 6), suggesting that failure to dephosphorylate these specific PA species might play a role in the altered kinetics of INO1 expression in the pah1Δ strain versus wild type (Fig. 2). In wild type cells, incorporation of label into these PA species was similar at 0 and 60 min after the media shift, followed by a decrease in label incorporated into PA species 32:1 and 34:2 at 180 min (Fig. 6). Strikingly, the label associated with PA species 34:1 increased significantly between 0 and 60 min following the shift to I − medium in both wild type and pah1Δ strains (Fig. 6). Moreover, the timing of the relative increase in deuterium label incorporated into PA species 34:1 correlates with the kinetics of INO1 expression in both wild type and pah1Δ following the shift to I − medium (Compare Figs. 2, A and B and 6).
Changes in the synthesis of PA species 32:1 and 34:1 after removal of inositol in the ole1 ts mutant relative to wildtype are correlated to inability to derepress INO1 The increase in the synthesis/retention of the PA species 34:1 observed in both wild type and pah1Δ (Fig. 6) between 0 and 60 min is correlated with the burst in INO1 expression that occurs after 60 min following removal of inositol (Fig. 2). PA species 34:1 contains either one C16:0 acyl chain and one C18:1 acyl chain or one C16:1 acyl chain and one C18:0 acyl chain. As Ole1 is the sole fatty acid desaturase in yeast (25), it is responsible for generating both the 16:1 and 18:1 unsaturated fatty acids. Thus, we reasoned that reduced production of these unsaturated fatty acids in the ole1 ts strain should result in diminished synthesis of the PA species 34:1 and, in turn, influence the kinetics of derepression of the INO1 gene. To test this hypothesis, we used a temperature sensitive strain, ole1 ts (26) to examine and compare the kinetics of synthesis of both PA species relative to the kinetics of increasing INO1 expression following the shift to I − medium. However, the growth of the ole1 ts mutant at 37 C is significantly reduced even in medium containing inositol, and cells of this strain died shortly after the shift to I − medium (data not shown). For this reason, experiments were carried out at the semipermissive temperature of 30 C, while maintaining cultures in logarithmic phase. Cells were labeled with deuterium-labeled glycerol for 25 min at the same time points as described above for wild type and pah1Δ. At time 0, the label incorporated into PA species 32:1 in the ole1 ts strain was half the value observed in wild type cultures under identical conditions (Fig. 6). The PA species that showed the greatest increase in labeling in ole1 ts under these circumstances was PA 32:1 (Fig. 6). This observation suggests that the ole1 ts mutant can increase synthesis of 16:1 and 18:1 if incubated for a longer period in I − medium at the semipermissive temperature of 30 C. Under these circumstances, the label associated with PA species 34:1 was 4-fold lower than the value detected in wildtype (Fig. 6). The amount of label associated with PA species 32:1, 34:1, and 34:2 in ole1 ts at 60 min after the media shift was like the levels found at zero time (Fig. 6). The increase in the label incorporated into these PA species at later time points (Fig. 6) could be due to breakdown of storage lipids, which can supply the demands for unsaturated fatty acids for a short period of time. Importantly however, INO1 was not expressed to any significant extent in the ole1 ts mutant after the shift to I − medium and increased by only 10-fold overall as compared to 350-to 400-fold in wild type (Fig. 7).

Discussion
Addition of inositol to actively dividing wild type cells adapted to growth in I − medium triggers a rapid decline in PA In the current study, we exploited the slower patterns of changes in both lipid metabolism and gene expression to gain insight into the potential roles of individual PA species in this complex metabolism and regulation. Following the shift of both wild type and pah1Δ cells to I − medium, overall PA levels rose with similar kinetics in the two strains (Fig. 2). However, derepression of INO1 was delayed in pah1Δ cells in comparison to wild type. This finding indicates that the activity of the Pah1 PA phosphatase is instrumental in controlling a pool of PA critical for the rapid derepression of INO1 in wild type cells following a shift to I − medium. In addition, in the course of ongoing metabolism, there are multiple sources that generate and utilize PA, thus having the potential to affect the expression of UAS INO -containing genes, especially INO1. PA is the central precursor in the synthesis of both membrane phospholipids and the major storage lipid TAG. In growing cells, PA is generated in an ongoing fashion by de novo from glycerol-3-P and acyl-CoA, but also through Dgk1 DAG kinase activity, which catalyzes the phosphorylation of DAG to PA (27,28). Phospholipase D-mediated turnover of PC also results in the production of PA and free choline (9). While phospholipase D activity is localized mainly in the plasma membrane and endosomes (29,30), the enzymes that catalyze the de novo synthesis of PA are found in the ER and lipid droplets (31,32). We favor the idea that INO1 derepression in the absence of inositol is responding to the pool of PA that is newly synthesized in the ER. Our reasoning is based in a previous study in which we demonstrated that upon addition of inositol into the growth medium, newly synthesized PA is rapidly depleted in the ER in direct response  to a rapid increase in PI synthesis (1). Our current analysis using deuterium-labeled glycerol is consistent with the hypothesis that a specific pool of newly synthesized PA precedes the burst in INO1 expression following inositol depletion. We observed that in wildtype cells, deuterium-labeled glycerol was incorporated preferentially into PA species 34:1 followed by PA species 32:1, 34:2 and 32:2 (Fig. 6). However, PA species 34:1 was the only molecular species of PA that exhibited an increase in the rate of synthesis from 0 to 60 min (Fig. 6), suggesting that the burst in INO1 expression that occurs from 60 to 120 min after the shift to I − medium may be responding to the abundance of this PA molecular species, as opposed to the overall cellular pool of PA. Incidentally, this molecular species is the major one found in wild type and pah1Δ cells (33). Importantly, the increase in the synthesis of the PA species 34:1 precedes the spike in INO1 expression, and this PA species must reach certain threshold to induce expression of INO1, which it is met or exceeded in the case of wildtype and pah1Δ but not in ole1 ts mutant. Previous studies have characterized the PA species profile of wild type and pah1Δ grown in YEPD medium (33) and in wildtype cells grown in medium containing different carbon sources (34). Indeed, the composition of the growth medium has a profound influence on the PA species profile, which exhibit a remarkably degree of variability (34). In the current study, we report the changes in the levels of PA species in wild type following a shift to medium lacking inositol (Fig. 3C). Since PA is the entry point to the phospholipid biosynthetic pathway, the relative amounts of the different PA molecular species depend on their utilization in the generation of lipids such as PI and PC (Fig. 3, A and C). It is interesting to note that once an acyl chain has been transferred from acyl-CoA to a PA backbone, it is no longer susceptible to elongation or desaturation (35). Consequently, PA can be seen as a moderator between the free acyl-CoA pool and the acyl-CoA bound to phospholipids.
The Opi1 repressor controls the expression of INO1 via the Opi1/Ino2-Ino4 (Henry) regulatory circuit (1-8) (Fig. 1). In the absence of inositol, Opi1 interacts with PA in the perinuclear ER, Opi1 contains a motif, FFAT (two phenylalanines in an acid tract), shown by Loewen et al. (2003) (36) to interact with an integral ER-associated protein Scs2 (Fig. 1). Since Opi1 tethering to the ER requires interaction with both Scs2 and PA, Opi1 remains in the ER when cells are growing in the absence of inositol, resulting in derepression of INO1 and coregulated UAS INO -containing genes (1) (Fig. 1). Both the headgroup and the acyl chains of PA are critical in the binding of Opi1 to the ER membrane. Kooijman and Burger (37) pointed out that unsaturated PA has a special cone shape that facilitates its interaction with proteins. To our knowledge, the present study has revealed for the first time that the PA species 34:1 may be involved in the recruitment of Opi1 to the ER in response to a shift to medium lacking inositol. However, in vitro liposome binding assays have shown that the Opi1 repressor exhibited higher binding affinity toward shorter-chain and moresaturated PA species (18). However, the relative specific affinity of the Opi1 repressor for the PA molecular species 34:1 has yet not been tested and compared to other PA species.
Moreover, liposome binding assays lack the influence of Scs2, which is required for the interaction of Opi1 to PA in vivo (1).
In summary, we have found that the kinetics of INO1 derepression in wild type cultures that have been shifted to medium depleted of inositol are influenced not only by the overall level of PA but also by the rate of PA synthesis. The synthesis of the PA species 34:1 appears to be critical for the induction of INO1 in the absence of inositol. Further studies are necessary to determine if this PA species interacts in vivo with the Opi1 repressor.

Culture conditions
Cultures were maintained on YEPD (1% yeast extract, 2% peptone, 2% glucose, 2% agar) media plates. All experiments were conducted using cultures grown to mid-logarithmic phase at 30 C on a rotary shaker (New Brunswick Scientific Co, Inc) at 200 rpm using chemically defined synthetic media as described by Jesch et al. (38). Cells were grown in 50 ml batches of complete synthetic media with (I + ) or without (I − ) inositol (75 μM) as indicated. Solid media had the same composition plus 2% agar.

Strains
Yeast strains used are listed in Table 1. Deletion mutant strains were generated by PCR-mediated gene replacement as described previously (40). The plasmid pRS315 was used as a template to generate a PCR fragment for the PAH1 and PCT1 gene disruption. The entire open reading frame of the PAH1 and PCT1 genes were replaced with the LEU2 marker gene. Leucine prototrophs were screened by colony PCR to verify integration at the correct genetic locus.

Materials
All chemicals were reagent grade. Phospholipid standards were purchased from Avanti Polar Lipids. Neutral lipid standards were purchased from Nu-Chek Prep. Deuterium-labeled glycerol (1,1,2,3,3-D5, 99%) was purchased from Cambridge Isotope Laboratories, Inc. [ 32 P]-orthophosphate was purchased from PerkinElmer, and scintillation-counting supplies were purchased from National Diagnostics. Silica gel loaded SG81 chromatography paper was purchased from Whatman, Inc, and HPTLC plates from Merck. Growth medium supplies were purchased from Difco Laboratories.

RNA isolation and RT-PCR analysis
To measure expression of the INO1 gene, cells were grown on medium containing inositol to mid-logarithmic phase at 30 C and allowed to grow to A 600 = 0.5 to 0.6. Cultures were filtered, washed with prewarmed medium lacking inositol, and resuspended in new medium lacking inositol, maintaining the same temperature. Samples were harvested by filtration at every hour for a period of 5 to 7 h following the media shift, flash-frozen in dry ice, and stored at −80 C.
Total RNA was isolated using RNeasy Mini Kit including a DNA digestion with RNase-free DNase Set (both Qiagen). In brief, the reaction mix in a volume of 25 μl consisted of 0.5 μM primers, 0.2 μM TaqMan probe, 1× master mix, and 5 ng cDNA. All reactions were performed in technical duplicate. Nontemplate control (5 ng RNA) and nonreaction control (DEPC-water) were routinely performed. The thermal program for the PCR included stage 1: 95 C, 10 min, stage 2: 95 C, 0.5 min, and 60 C, 1 min for a total of 40 cycles, and stage 3: hold at 4 C. Relative quantitation was done using the ΔΔCt method (see StepOnePlus user manual of Applied Biosystems). The ΔΔCt represents the change in mRNA expression after ACT1 normalization relative to the wildtype control calculated as 2 −(Gene Ct , where "gene" represents the mRNA under study (INO1), "x" refers to the strain from which the mRNA to be tested was derived (i.e., wildtype or quadruple mutant), and "cr" refers to the control mRNA. The value "cr" (for control mRNA) was derived from level of mRNA in the wildtype strain, pregrown as described above in I + medium at 30 C, but shifted to fresh medium of the same composition (i.e., containing inositol) at the same temperature. The "Ct" (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., to exceed background level). Each RT-PCR experiment was performed at least in triplicate.

Kinetic analysis of changes in PA composition following inositol addition
To determine changes in the composition of PA over a set time course following inositol depletion, cells were grown in I + medium, and phospholipids were labeled with 20 μCi/ml [ 32 P]orthophosphate (specific activity of isotope was 2.7 mCi/mmole phosphate) to steady-state. When the cells reached the mid-logarithmic phase of growth (A 600 = 0.5) were filtered, washed with prewarmed medium lacking inositol, and resuspended in I − medium maintaining constant the specific activity of the label. Cultures were incubated at 30 C for additional 300 min. Five milliliters samples were taken at 0, 60, 120, 180, 240, and 300 min and were mixed with 0.5 ml 50% trichloroacetic acid (TCA) and allowed to stand on ice for 20 min. The samples were washed twice with distilled water. Labeled lipids were extracted as previously described (41). The individual phospholipids species were resolved by twodimensional paper chromatography (42). Phospholipid identity was based on the mobility of known standards and quantified on a STORM 860 PhosphorImager (Amersham Biosciences).

Analysis of PA synthesis following inositol deprivation
To determine changes in the synthesis of PA over a set time course following inositol starvation, cells were pregrown in I + medium until mid-logarithmic phase of growth (A 600 = 0.5). At this time point, cells were filtered, washed with prewarmed medium lacking inositol and resuspended in I − medium at 30 C. 100 μCi/ml [ 32 P]-orthophosphate (specific activity of isotope was 13.51 mCi/mmole phosphate) was immediately added to the cultures shifted to I − medium, and these cells were harvested 20 min later. This sample is referred to as the 0-time point. 32 P was added in similar fashion to cultures at 60, 180, and 300 min following the media shift. For each time point, samples were harvested 20 min after addition of label. Five milliliters samples were mixed with 0.5 ml 50% TCA and allowed to stand on ice for 20 min. The samples were washed twice with distilled water. Lipids were extracted and analyzed as described above for 32 P steady-state labeling.

Electrospray ionization mass spectrometry analysis of lipids
To analyze the phospholipid and neutral lipid species following a shift to medium lacking inositol, cells were pregrown in I + medium until mid-logarithmic phase of growth (A 600 = 0.5). The cells were then shifted as described above to medium lacking inositol and incubated for additional 300 min. Two sets of samples (A 600 = 25), one set to be used for phospholipid extraction and the other set to be used for MATα ole1 his7Δ lys2Δ ura1Δ tyr1Δ gal1Δ ade1Δ ade2Δ (26) neutral lipid extraction, were collected at 0, 60, 180, and 300 min by rapid filtration through Durapore membrane filters (0.65 μm DVPP. Millipore, Inc) and immediately transferred to a tube containing 5 ml of 10% TCA in an ice bath for 25 min. The cell membranes were spun down, and the supernatant discarded. The pellet was washed twice with water. The cell membrane phospholipids were extracted with 1 ml of a solvent mixture consisting of ethanol, water, ethyl ether, pyridine, and ammonium hydroxide (45:45:15:3:0.003, v/v/v/v/v). Fifty microliter of 0.05 mg/ml phospholipid internal standards (PA 28:0, PC 34:0 and PI from bovine liver) were added to the extraction mixture. Samples were sonicated for 20 min and incubated at 60 C for 50 min. Cell debris were pelleted by centrifugation at 5000g for 10 min, and the supernatant was collected. The supernatant was dried under a stream of nitrogen. The lipid film was resuspended in 300 μl of chloroform: methanol 1:1 v/v for MS analysis. The cell neutral lipids were extracted with a mixture of chloroform: methanol 2:1 v/v. 50 μl of 0.05 mg/ml neutral lipid internal standards (DAG 28:0 and TAG 51:0) were added to the extraction mixture. Samples were sonicated for 20 min and incubated at room temperature for 60 min. The chloroform phase was dried, and the residue was dissolved in 300 μl of chloroform: methanol 1:1 v/v for MS analysis.
Quantitative analysis of polar and neutral lipid species was performed using a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific) configured for microbore and outfitted with fused silica transfer lines coupled with an AB/ Sciex 4000 Q Trap mass spectrometer (Applied Biosystems) fitted with a Turbo Ion Spray source operated in multiple reaction monitoring (MRM) mode. In MRM experiments, it was provided a unit resolution for both Q1 and Q3, 50 ms dwell time for each transition ion pair and Q1 and Q3 MRM transition pairs. The standards PA 28:0, PC 34:0, DAG 28:0, TAG 51:0, and PI from bovine liver were diluted to 20 ng/μl and infused at 5 μl/min directly into the mass spectrometer and relevant MRM parameters optimized using parameter ramping following manufacturer's recommended protocol. PA and PI lipid species were optimized for acquisition in negative ion mode as deprotonated adducts [M − H] − , PC lipid species were optimized for acquisition in positive ion mode as protonated adducts [M + H] + , DAG, and TAG lipid species were optimized for acquisition in positive ion mode as lithium adducts [M + Li] + . Samples for PA and PI species analysis were diluted 1:20 with eluent A in glass insert autosampler vials (SUN-SRi) with an injection volume of 5 μl using a user defined program with eluent A in pick-up vials 1 and 2 and infused at a rate of 65 μl/min and separated on a Viva Silica 5μ, 300A, 200 × 1 mm (Restek) column at 30 C. The separation was carried out using a linear gradient as follows: 0-2-20-24-25-38 min, 10-10-100-100-10-10 % eluent B, mass spectrometer data acquisition start trigger at 2 min [Eluent A: 58/40/2 isopropanol/hexane/water (v/v/v) with 0.2% glacial acetic acid (v/v) and 50 mM ammonium acetate (w/v); eluent B: 50/40/10 isopropanol/hexane/water (v/v/v)] with 0.2% glacial acetic acid (v/v) and 50 mM ammonium acetate (w/v)]. Samples for PC species analysis were treated as above and analyzed using a slightly different linear gradient as follows: 0-1-32-35-36-48 min, 5-5-85-85-5-5 % eluent B, mass spectrometer data acquisition start trigger at 2 min and an injection volume of 2 μl using user defined program with eluent A in pick-up vials 1 and 2. The samples for DAG and TAG species analysis were diluted 1:10 (v/v) with eluent A in glass insert autosampler vials with an injection volume of 10 μl using user defined program with eluent A in pick-up vials 1 and 2 and infused at a rate of 60 μl/min and separated on an Ultra C1 3, 100A, 150 × 1 mm (Restek. Bellefonte, PA) column at 30 C. The separation was carried out using a linear gradient as follows: 01.5-2-30-31-36-37-52 min, 0-0-5-75-100-100-0-0 % eluent B, mass spectrometer data acquisition start trigger at 1 min [Eluent A: 60/20/20 methanol/ isopropanol/water (v/v/v) with 0.2% glacial acetic acid (v/v), 50 mM ammonium acetate (w/v), and 2 mM lithium acetate (w/v); Eluent B: 5/90/5 methanol/isopropanol/water (v/v/v) with 0.2% glacial acetic acid (v/v), 50 mM ammonium acetate (w/v), and 2 mM lithium acetate (w/v)]. Raw data were acquired using Analyst, version 1.6.1 (AB/Sciex). and raw MRM data were processed using MultiQuant, version 2.1.1 (AB/Sciex).

Short-time labeling with deuterium-labeled glycerol
Cells grown to mid-logarithmic phase in I + medium were collected by filtration, washed with prewarmed medium lacking inositol, and resuspended in I − medium at 30 C. A 25 ml sample was immediately transferred to a different flask and supplemented with 50 mM deuterium-labeled glycerol (1,1,2,3,3-D5, 99%). These cells were harvested 25 min later. This sample is referred to as the zero-time point. Deuteriumlabeled glycerol was added in similar fashion to cultures at 60, 180, and 300 min after the shift to I − medium. For each time point, 25 ml samples were harvested by filtration 25 min after addition of the deuterium-labeled glycerol, immediately mixed with 5 ml 5% TCA and allowed to stand on ice for 20 min. The samples were washed twice with distilled water. Lipids were extracted and analyzed as described above for the mass spectrometry analysis of lipids.

Data availability
All the data produced for this work are contained within the article.
Acknowledgments-We thank George Carman for helpful comments during this work.